The present invention pertains to a high-yield, dual-buffered growth medium that supports the growth of bacterial cell cultures at higher densities than those achieved by conventional rich media.
The propagation of recombinant DNA in E. coli is a ubiquitous and necessary prerequisite for almost any endeavor in molecular biology, from cloning and sequencing of genes to ectopic expression of proteins. Many vectors have been used for this purpose, including plasmids, cosmids, and bacterial artificial chromosomes (BACs). Rich media such as Luria-Bertani (LB) broth, xe2x80x9cSuperbroth,xe2x80x9d and xe2x80x9cTerrific Brothxe2x80x9d (TB) have been developed to enhance the normal growth of transformed bacterial cells.
See generally, F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, pp. 1.1.1 -1.1.3 (1997); J. Sambrook, E. F. Fritsch, and R. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., CSH Laboratory Press, pp. 1.21-1.23, A1-A3 (1989); K. D. Tartof and C. A. Hobbs, xe2x80x9cImproved Media for Growing Plasmid and Cosmid Clones,xe2x80x9d Bethesda Res. Lab. Focus vol. 9, p. 12 (1987). LB and its derivatives are the most commonly used, possibly because more complex recipes improve yield only slightly. In most cases, the moderate increase in yield does not justify the added time and effort to make the more complex media.
Cell density in liquid culture is limited by certain byproducts of metabolism. Growth of E. coli on excess glucose under aerobic conditions causes formation of acidic by-products, of which acetate is the most predominant. See H. W. Doelle et al., xe2x80x9cRegulation of glucose metabolism in bacterial systems,xe2x80x9d Adv. Biochem. Eng. vol. 23, pp.1-35 (1982); G. W. Luli and W. R. Strohl, xe2x80x9cComparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations,xe2x80x9d Appl. Environ. Microbiol. vol. 56, pp. 1004-1011 (1990)). High acetate concentrations can severely inhibit cell growth, genetic stability, and recombinant protein production. (G. W. Luli and W. R. Strohl, Appl. Environ. Microbiol. vol. 56, pp. 1004-1011 (1990)). While high cell density techniques have been developed for fed-batch fermentations (reviewed in G. L. Kleman and W. R. Strohl, xe2x80x9cDevelopments in high cell density and high productivity microbial fermentation,xe2x80x9d Current Opinion in Biotechnology vol. 5 pp. 180-186 (1994)), none have been developed for small-scale applications appropriate for the typical research laboratory.
U.S. Pat. Nos. 4,656,132 and 4,894,334 disclose a process for improving the yield of heterologous protein produced by recombinant bacteria by supplementing the nutrient medium with a water soluble alcohol and an amino acid mixture during the terminal phase of cultivation.
U.S. Pat. No. 5,223,418 discloses a method of improving the yield of heterologous proteins produced by recombinant Streptomyces lividans by using a complex nutrient medium supplemented with high concentrations of casamino acids (1-5%) and glucose (up to 3%).
U.S. Pat. No. 5,658,790 discloses a method to prepare a cell culture medium by using unit dose packaging of a dry concentrate of the medium. The formulations contain a single buffer, Tris, to maintain a neutral pH. Alternative embodiments contain low concentrations of glucose (0.4%) and casamino acids (0.6%).
In addition to external sources of carbon, nitrogen, and energy, bacteria can utilize an external source of ribonucleosides. Ribonucleotides in the growth medium can be hydrolyzed by periplasmic phosphatases, generating permeable ribonucleosides that can enter the cell and be used for RNA synthesis. (H. Zimmerman, xe2x80x9c5xe2x80x2-Nucleotidase: Molecular Structure and Functional Aspects,xe2x80x9d Biochemical Journal vol. 285, pp. 345-365 (1992)).
No known prior culture medium has a dual-buffer system or adds a source of free ribonucleotides.
There is a long felt need by those practicing recombinant DNA techniques for a high efficiency, inexpensive growth medium that can support high bacterial cell densities and high yields of plasmid DNA or of recombinant protein using equipment available in an ordinary research laboratory.
We have discovered a novel growth medium that supports much higher bacterial cell densities and, concomitantly, a much higher yield of plasmid than has been previously reported for small-scale applications. The high-yield growth medium contains two buffers with different pKa values and optionally includes an additional nucleotide source. On a unit volume basis, E. coli cultures grown in this medium have consistently produced 5-10 times, and sometimes up to 30 times, more recombinant plasmid than was produced in conventional rich media, paralleling the increase in cell density. This phenomenon is independent of E. coli host strain, DNA insert size, and plasmid copy number. The medium is economical, and high yields can be achieved using standard research laboratory equipment.